Difference between revisions of "Part:BBa K1694014"
 
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[[File:EG14.png|200px|thumb|right|'''Fig.1''' OmpA-N-scFv(anti-EGFR)]]
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[[File:EG14.png|200px|thumb|right|'''Fig.1''' OmpA-N-scFv(Anti-EGFR)]]
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To display the antibody outside the ''E. coli'', we used Lipoprotein-Outer membrane protein A (Lpp-OmpA). According to the paper reference, We chose the first 9 amino acid of Lpp, and the 46~159 amino acid of OmpA.
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To display <html><a href="https://parts.igem.org/Part:BBa_K1694004">scFv</a></html> outside the ''E. coli'', we used <html><a href="https://parts.igem.org/Part:BBa_K1694002">Lipoprotein-Outer membrane protein A</a></html> (Lpp-OmpA). According to the paper reference, we chose the first 9 amino acid of Lpp and the 46~159 amino acid of OmpA. When designing ''XbaI-SpeI'' restriction site between Lpp-OmpA and scFv, it will cause a mixed site. Therefore, the ''NcoI'' restriction site rather than the EX-SP restriction site was designed.
 
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In order to change the scFv parts easily, we added a ''NcoI'' restriction site between OmpA and scFv so that we can change various scFv DNA sequence by using the ''NcoI'' restriction enzyme.<br>
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In order to easily change various scFv DNA sequence, we added a ''NcoI'' restriction site between OmpA and scFv.<br>
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Fig.2 showed how we combine Lpp-OmpA-N and scFv together. First, we use restriction enzyme ''NcoI'' to digest the upstream and downstream parts. After ligate two digest product, there are no mixed site in Lpp-OmpA-N-scFv.
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The Fig.2 showed how we design Lpp-OmpA-N and scFv into ''NcoI'' restriction site. First, we digest Lpp-OmpA part with ''EcoRI'' and ''NcoI'', scFv part with ''NcoI'' and ''PstI.'' Then we ligated them together.  
  
 
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[[File:131415.png|600px|thumb|left|'''Fig.2''' The combination of OmpA-N-scFv]]
 
[[File:131415.png|600px|thumb|left|'''Fig.2''' The combination of OmpA-N-scFv]]
 
[[File:ompascfv.png|250px|thumb|left|'''Fig.3''' OmpA-N-scFv]]
 
[[File:ompascfv.png|250px|thumb|left|'''Fig.3''' OmpA-N-scFv]]
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<html>
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Introduction of basic parts:
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<br>
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<a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1694004"> Anti-EGFR </a><br>
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</html>
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<h1>'''Experiment:'''</h1>
 
<h1>'''Experiment:'''</h1>
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[[File:OC.png|200px|thumb|left|'''Fig.4''' The PCR result of the Lpp-OmpA-N+scFv. The DNA sequence length is around 1000~1200 bp, so the PCR products should appear at 1200~1400 bp.]]  
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[[File:OC.png|200px|thumb|left|'''Fig.4''' The PCR result of the Lpp-OmpA-N+scFv. The length of DNA sequence is 1181 bp, so the PCR products should appear at 1200~1400 bp.]]  
  
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After receiving the DNA sequences from the gene synthesis company, we recombined each OmpA-N-scFv gene to PSB1C3 backbones and conducted a PCR experiment to check the size of each OmpA-N-scFv. The DNA sequence length of the OmpA-N-scFv are around 1000~1200 bp. In this PCR experiment, the OmpA-N-scFv products size should be near at 1200~1400 bp. The Fig.4 showed the correct size of the scFv, and proved that we successful ligated the scFv sequence onto an ideal backbone.
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After receiving the DNA sequences from the gene synthesis company, we recombined each OmpA-N-scFv gene to pSB1C3 backbones and conducted a PCR experiment to check the size of each OmpA-N-scFv. The length of OmpA-N-scFv DNA sequence is 1181 bp. In this PCR experiment, OmpA-N-scFv products size should be near at 1200~1400 bp. The Fig.4 showed the correct size of the scFv, and proved that we successfully ligated the scFv sequence onto an ideal backbone.
  
  

Latest revision as of 07:48, 22 September 2015

OmpA-scFv(Anti-EGFR)

Introduction:


Fig.1 OmpA-N-scFv(Anti-EGFR)

To display scFv outside the E. coli, we used Lipoprotein-Outer membrane protein A (Lpp-OmpA). According to the paper reference, we chose the first 9 amino acid of Lpp and the 46~159 amino acid of OmpA. When designing XbaI-SpeI restriction site between Lpp-OmpA and scFv, it will cause a mixed site. Therefore, the NcoI restriction site rather than the EX-SP restriction site was designed.
In order to easily change various scFv DNA sequence, we added a NcoI restriction site between OmpA and scFv.
The Fig.2 showed how we design Lpp-OmpA-N and scFv into NcoI restriction site. First, we digest Lpp-OmpA part with EcoRI and NcoI, scFv part with NcoI and PstI. Then we ligated them together.


Fig.2 The combination of OmpA-N-scFv
Fig.3 OmpA-N-scFv


Introduction of basic parts:
Anti-EGFR


Experiment:

Fig.4 The PCR result of the Lpp-OmpA-N+scFv. The length of DNA sequence is 1181 bp, so the PCR products should appear at 1200~1400 bp.

After receiving the DNA sequences from the gene synthesis company, we recombined each OmpA-N-scFv gene to pSB1C3 backbones and conducted a PCR experiment to check the size of each OmpA-N-scFv. The length of OmpA-N-scFv DNA sequence is 1181 bp. In this PCR experiment, OmpA-N-scFv products size should be near at 1200~1400 bp. The Fig.4 showed the correct size of the scFv, and proved that we successfully ligated the scFv sequence onto an ideal backbone.


Fig.5 OmpA-N-scFv(Anti-EGFR)

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 388
  • 1000
    COMPATIBLE WITH RFC[1000]